Abstract Pancreatic cancer remains a deadly cancer with a 5-year survival rate of 8%. The development of targeted therapies to proteins that are regulated by mutant Kras (present in >90% of pancreatic cancers) could improve the response to pancreatic cancer therapy such as gemcitabine. NADK is an enzyme that converts NAD+ into NADP+, which is used by malic enzyme (ME1) to produce pyruvate and NADPH from malate in the glutamine reprogramming pathway (GRP). NADPH is a coenzyme in metabolism pathways and reduces oxidative stress, which can affect the response to oxidative stress inducing chemotherapeutic agents such as gemcitabine. Inhibition of NADK in gemcitabine treated pancreatic cancer cells may increase its efficacy because NADK inhibition induces ROS that can push oxidative stress levels past a toxic threshold. Our preliminary data show that knockdown of NADK significantly inhibited pancreatic tumor growth and increased ROS levels in AsPC1 tumor xenografts in mice. Oxidative stress activates NRF2, a master regulator of redox (e.g. the thioredoxin and glutathione systems) and phase II response associated transcription that is activated by mutant Kras and implicated in gemcitabine resistance. Overoxidation of the NRF2 regulated thioredoxin and glutathione systems could result in the induction of apoptosis due to a reduced capacity to reduce oxidized proteins when NADK is inhibited. NRF2 is also induced in response to conditioned media from pancreatic stellate cells, which regulates proliferation of pancreatic cancer cells and expression of metabolic genes. Understanding how NADK inhibition affects NRF2 signaling in fibrotic pancreatic cancer tissue will be essential to determining the mechanisms by which NADK-induced oxidative stress affects pancreatic tumorigenesis and the response to gemcitabine. Our goal in the parent grant is to investigate the ability of NADK inhibition to induce oxidative stress and apoptosis in gemcitabine treated pancreatic cancer tissue. In this supplemental application, we will determine how NADK inhibition affects the redox associated signaling pathways that will mediate the response to NADK inhibition and gemcitabine in pancreatic cancer. We hypothesize that the inhibition of NADK will disrupt the function of NRF2 and thioredoxin system redox signaling networks and enhance the effectiveness of gemcitabine treatment in pancreatic cancer. We will investigate our hypothesis with the following specific aims: Aim 1- Determine the role of NRF2 in mediating the response to oxidative stress induced by NADK inhibition and Aim 2- Determine the effect of NADK inhibition on the function of the thioredoxin system and its role in apoptosis. Understanding how NRF2 and thioredoxin system signaling are affected by fibrosis and the inhibition of NADK in pancreatic cancer will be essential to improving the efficacy of gemcitabine treatment. The defined role for NRF2 in the regulation of antioxidant protein expression and its emerging role in chemoresistance to gemcitabine in pancreatic cancer lay the foundation for defining the mechanisms by which NADK inhibition induces ROS induced-apoptosis as proposed in the parent R01 and further supported in this supplemental application.